Osterix (Osx) is an osteoblast-specific transcription factor and is required for bone formation and maintenance of bone homeostasis in adult life. It activates a repertoire of genes essential for maturation and function of osteoblasts during bone formation. This study focuses on epigenetic phenomena that control the activation of bone-specific genes in parallel to the transcriptional activity of Osterix during skeletal formation. This proposal addresses the core mission of funding agency NIAMS which promotes research funding in our understanding of bone and bone related diseases. The proposal has three specific aims; each one addresses a specific question to better understand the transcriptional control of bone-specific genes by the master regulator Osx in osteoblasts. Specific aim 1 is to test our hypothesis that the chromatin of Osx-target genes is under epigenetic control of histone methylation and demethylation through NO66 demethylase during osteoblast differentiation. Chromatin immunoprecipitations (ChIP) assays using specific antibody will be performed to determine the interaction sites of Osx and NO66, and the levels of histone methylation of H3K4me3 and H3K36me3 at Osx target genes. Mouse calvarial osteoblasts, isolated at embryonic stages E14.5 and E18.5, and postnatal day 7 (P7) of bone formation, will be used for immunoprecipitation with antibodies followed by hybridization of immunoprecipitated DNA to a high density custom DNA array containing several osteoblast genes. This method is called ChIP-on-ChIP. Next, expression profiles of Osx target genes will be measured at different stages and correlated with the dynamics of the interactions of Osx and NO66, and the levels of active histone methyl marks H3K4me3 and H3K36me3 at those Osx target genes. Specific aim 2 is to test whether the DNA of Osx target genes are methylated on CpG dinucleotides prior to their activation. The CpG methylation sites will be first identified in the promoters of the Osx target genes including Bsp, Oc, and Sost by bisulfite treatment to DNA followed by PCR amplification and sequencing. Sodium bisulfite converts all unmethylated cytosine in DNA to uracil, but not the methylated cytosine. Genomic DNA isolated from Osx null calvarial cells will be used to first identify CpG methylation in the DNA of the promoter of Osx target genes which are activated by Osx. Next we will study whether there is an inverse relationship between the DNA methylation of Osx target promoters and the transcriptional state of the respective genes at different stages (E13.5, E15.5, E18.5 and postnatal day 7) during bone formation in wild type mice. Finally specific aim 3 is to study the potential biological significance of methylation of Osx in osteoblasts. Using a mass spectrometry analysis of Osx polypeptides, several lysine residues of Osx were identified as post-translationally modified by methylation. Biochemical and in vitro studies will be performed to study the potential role of Osx methylation in activation of Osx target genes during osteoblast differentiation.